Study On Optimization Of Fermentation Process Of Grifola Frondosa Laccase And Its Synergistic Degradation Of Lignin By Probiotics

My country can produce about 900 million tons of lignin wastes（such as straw,etc.）each year.Because they are not effectively used,they not only cause waste of resources,but also cause many severe environmental problems.Due to the complexity of the structure of lignin,its efficient use is greatly restricted.Microbial degradation of lignin has the advantages of greenness,safety,specificity and sustainability,and has been widely used.Lignin degrading enzyme is a kind of biological enzyme released to the outside of the cell by microorganisms,which has a strong effect of catalyzing oxidative degradation of lignin.Studies have found that laccase can selectively catalyze and degrade lignin without producing toxic substances,and the reaction conditions are mild,low cost,and easy to implement.Among them,basidiomycetes can use their own enzyme system to degrade lignin,and Grifola frondosa,as a kind of basidiomycetes,is a rare and medicinal mushroom that can produce a large amount of laccase,but it is rarely studied in feed production.In this experiment,the laccase produced by Grifola frondosa by solid-state fermentation of bran was studied,and the biomass,mycelial morphology,and laccase activity of Grifola frondosa were analyzed.The enzymatic characteristics of Grifola frondosa laccase were extracted and analyzed.The effect of flower laccase on the lignin content and structure of melon seed peel,corn stover,korshinskii and corn cob fermented by probiotics.The mechanism of laccase synergistic fermentation with probiotics to degrade lignin was preliminarily discussed,which is the development and utilization of Grifola frondosa laccase Laid the theoretical foundation.The thesis mainly includes the following three parts.（1）The effect of bran on the fermentation ability of Grifola frondosa to produce laccaseUsing biomass,mycelial morphology and laccase activity as indicators,the growth,morphology and laccase production ability of Grifola frondosa in PDB medium,bran liquid medium and bran solid medium were studied.The results showed that the biomass of Grifola frondosa mycelium in the PDB medium reached 0.11 mg/g on the 5th day,and the hyphae grew to the 5th day and showed the same size and uniformity of the mycelium.The color was white and flawless,and it began to age on the 7th day.Mass,yellowish color,laccase activity reached 73.24 U/g on the 5th day;in the bran liquid medium,the Grifola frondosa biomass reached 0.03 mg/g on the 9th day,and the mycelium gradually increased on the 9th day Expanded,the color is white,the color begins to age on the 13th day,and the fermentation liquid is turbid,and the laccase activity reaches 43.06 U/g on the 11th day;in the bran solid medium,the Grifola frondosa biomass reaches 0.04 mg/g on the 13th day g.Mycelium begins to expand on the 9th-11th day,white and dense,the surface of bran is completely covered,and ages on the 13th day,the hypha becomes yellow,the surface is smooth,the hypha is autolyzed,water droplets are produced,and laccase The vitality reached 170.32 U/g on the 13th day.Compared with liquid fermentation,the activity of solid fermentation laccase is significantly improved.In summary,in the later test,the fermentation time was selected as 13 days as the best fermentation time,and then the optimal process for the production of laccase by solid-state fermentation of Grifola frondosa from bran was screened,that is,the water content is 80%and the bran addition amount 70%,oak sawdust added 25.2%.Under these conditions,the laccase activity reached 179.72 U/g.（2）Study on the separation and enzymatic characteristics of Grifola frondosa laccaseThe crude laccase extract was extracted by the ammonium sulfate precipitation method,and the laccase activity was used as an indicator to study the effects of ammonium sulfate concentration,extraction time and extraction temperature on it in turn.Studies have shown that the best extraction conditions for Grifola frondosa laccase are ammonium sulfate concentration of 70%,time of 1 h,and temperature of 40℃.Under these conditions,the laccase activity reaches 193.84 U/g.Adding Cu²⁺will increase the activity of Grifola frondosa laccase,which can reach 207.36 U/g at 40℃and p H 5.4.（3）Study on the degradation of lignin by laccase synergistically with probioticsUnder the synergistic effect of laccase,it can increase the degradation rate of lignin in melon seed husk,corn stover,korshinskii and corn cob fermented by probiotics,while the degradation rate of corn cob is relatively high;through SEM technology,it can be concluded that the laccase synergy Under the action,the surface morphology and structure of melon seed skins,corn stalks,caragana korshinskii and corn cobs are changed,cracks appear and gaps become larger;Through FTIR analysis,it is concluded that the benzene ring absorption peak intensity of lignin characteristic absorption peaks of purple butyl and guaiacyl is reduced,and the structure of lignin is destroyed.Therefore,laccase can promote the basic structure of lignin,purple butyl and guaiac.Destruction of the benzene ring structure.In summary,Grifola frondosa solid-state fermentation of bran can produce laccase,and laccase synergistic fermentation with probiotics can reduce the lignin content of melon seed husks,corn stalks,caragana korshinskii and corn cobs,increase lignin degradation rate,and become a feed resource.Efficient use provides a beneficial reference.